Article Figures & Data
Figures
- Figure 1.
Trametinib and combination decrease T-lymphocyte proliferation. The proliferation index (A and C) and viability (B and D) of CD3/CD28-activated CFSE-labeled CD4+ (A and C) and CD8+ (B and D) T cells treated for 5 days with dabrafenib or trametinib, alone or in combination. Each point represents the mean value from the results obtained with 4 healthy donors; error bars show the SEM. One-way ANOVA with a Dunnett post test against the untreated control was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The proliferation index was determined using the proliferation tool in the FlowJo software package. This value represents the average number of cell divisions that the cells underwent by considering only the fraction of proliferated cells. Dabrafenib, BRAFi; trametinib, MEKi. Representative flow cytometry data are presented in Supplementary Fig. S1.
- Figure 2.
Trametinib and combination decrease T-lymphocyte cytokine production. The percentage of IFN-γ (A) and IL-17 (B) positive CD4+ T cells, and IFN-γ (C) and TNF-α (D) positive CD8+ T cells following treatment with dabrafenib or trametinib, alone or in combination was assessed by ICS flow cytometry 24 hours after activation by PMA–ionomycin. The percentage of cytokine-producing cells is shown as a fraction of total CD3+CD4+ (A and B) or CD3+CD8+ (C and D) T cells. Mean values with SEM from 8 healthy donors are shown. One-way ANOVA with a Dunnett post test against the untreated control was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dabrafenib, BRAFi; trametinib, MEKi. Representative flow cytometry data are presented in Supplementary Fig. S2.
- Figure 3.
Trametinib and combination affect the proliferation of antigen-specific T lymphocytes. The effect of kinase inhibition on in vitro stimulation of peptide-specific CD8+T cells was assessed 10 days after stimulation of PBMCs with an HLA-A2–restricted synthetic peptide for EBV BMLF1 (280–288). The percentage of dead PBMCs (A) and percentage of CD3+CD8+ T cells in the viable PBMC population (B) following 10 days of kinase inhibition are shown. Specific T cells were detected by ICS using fluorescently tagged antibodies to TNF-α (C) and IFN-γ (D). The percentage of TNF-α or IFN-γ producing CD8+ T cells was determined by fluorescence-activated cell sorting analysis. Each point represents a mean value from multiple stimulations performed with samples from 6 healthy donors and the mean with SEM is indicated. Two-way ANOVA with a Bonferroni post test against the DMSO control was performed. *, P < 0.05; ***, P < 0.001. Dabrafenib, BRAFi; trametinib, MEKi. Representative flow cytometry data are presented in Supplementary Fig. S3.
- Figure 4.
Dabrafenib and trametinib in combination inhibit cross-presentation in vitro. Immature HLA-specific moDCs were treated for 18 hours with dabrafenib or trametinib, alone or in combination in the presence or absence of NY-ESO-1 peptide (positive control) and NY-ESO-1 protein and His-tag antibody. After 24 hours, an NY-ESO-1–specific, HLA-restricted CD8+ T-cell clone was added to the treated and peptide-pulsed moDC to assess cross-presentation of NY-ESO-1 epitope in a standard recognition assay (no drug was present during the 4-hour APC–T-cell coculture) measuring specific cytokine secretion by the T-cell clone upon activation. Non–antigen-pulsed moDCs were used as the negative control (not shown). A, the capacity of HLA-A2+ moDCs to cross-present NY-ESO-1–derived epitope (157–165) to the NY-ESO-1–specific CD8+ T-cell clone (157-165/HLA-A2) in vitro. B, positive control; following drug treatment, moDCs were pulsed for 1 hour with the synthetic peptide for which the T-cell clone was specific. C, NY-ESO-1 protein with anti-IgG1 isotype control. Mean values with SEM from 4 healthy donors are shown. One-way ANOVA with a Dunnett post test against the control was performed; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dabrafenib, BRAFi; trametinib, MEKi.
- Figure 5.
Trametinib and combination promote moDC maturation and decrease moDC viability. A, moDCs were incubated with the indicated concentrations of individual inhibitors or combinations, for 24 hours, in the presence of LPS. Cells were stained with fluorescently labeled live/dead stain, CD86, CD83, or anti–HLA-DR antibodies. MFI of bound antibody was determined by flow cytometry. Representative single-parameter histograms show the expression of CD80, CD83, and CD86 by MoDCs (gray) after the addition of LPS (dotted black line gray) or 30 nmol/L trametinib plus LPS (solid black line). B, moDCs were incubated with the indicated concentrations of individual inhibitors or combinations, for 24 or 48 hours. The toxicity exerted by the individual and combined inhibitors was assessed using a fluorescent live/dead dye. Mean values with SEM from 4 healthy donors are shown. One-way ANOVA with a Dunnett post test against the control was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dabrafenib, BRAFi; trametinib, MEKi.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legends - PDF file - 79K
- Supplementary Figures 1 - 3 - PDF file - 376K, Supplementary Figure 1. Representative flow cytometry data of T-cell proliferation. Supplementary Figure 2. Gating strategy for T-lymphocyte cytokine production. Supplementary Figure 3. Gating strategy for antigen specific T-lymphocytes.
Volume 2, Issue 4
